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- EMDB-10219: The VgrG spike from the Type 6 secretion system -

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Basic information

Entry
Database: EMDB / ID: EMD-10219
TitleThe VgrG spike from the Type 6 secretion system
Map dataSharpened map
Sample
  • Complex: Tle1 effector bound to the VgrG spike of the type 6 secretion system
    • Protein or peptide: Putative type VI secretion proteinType VI secretion system
Function / homology
Function and homology information


Type VI secretion system spike protein VgrG2, C-terminal domain of unknown function DUF2345 / Putative type VI secretion system, Rhs element associated Vgr domain / Uncharacterized protein conserved in bacteria (DUF2345) / Putative type VI secretion system Rhs element Vgr / Type VI secretion system, RhsGE-associated Vgr family subset / Phage tail baseplate hub (GPD) / Type VI secretion system, RhsGE-associated Vgr protein / Gp5/Type VI secretion system Vgr protein, OB-fold domain / Type VI secretion system/phage-baseplate injector OB domain / Vgr protein, OB-fold domain superfamily
Similarity search - Domain/homology
Putative type VI secretion protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsRapisarda C / Fronzes R
Funding support France, Germany, 2 items
OrganizationGrant numberCountry
French National Research Agency France
European Communitys Seventh Framework Programme653706 Germany
CitationJournal: EMBO J / Year: 2020
Title: Structural basis for loading and inhibition of a bacterial T6SS phospholipase effector by the VgrG spike.
Authors: Nicolas Flaugnatti / Chiara Rapisarda / Martial Rey / Solène G Beauvois / Viet Anh Nguyen / Stéphane Canaan / Eric Durand / Julia Chamot-Rooke / Eric Cascales / Rémi Fronzes / Laure Journet /
Abstract: The bacterial type VI secretion system (T6SS) is a macromolecular machine that injects effectors into prokaryotic and eukaryotic cells. The mode of action of the T6SS is similar to contractile phages: ...The bacterial type VI secretion system (T6SS) is a macromolecular machine that injects effectors into prokaryotic and eukaryotic cells. The mode of action of the T6SS is similar to contractile phages: the contraction of a sheath structure pushes a tube topped by a spike into target cells. Effectors are loaded onto the spike or confined into the tube. In enteroaggregative Escherichia coli, the Tle1 phospholipase binds the C-terminal extension of the VgrG trimeric spike. Here, we purify the VgrG-Tle1 complex and show that a VgrG trimer binds three Tle1 monomers and inhibits their activity. Using covalent cross-linking coupled to high-resolution mass spectrometry, we provide information on the sites of contact and further identify the requirement for a Tle1 N-terminal secretion sequence in complex formation. Finally, we report the 2.6-Å-resolution cryo-electron microscopy tri-dimensional structure of the (VgrG) -(Tle1) complex revealing how the effector binds its cargo, and how VgrG inhibits Tle1 phospholipase activity. The inhibition of Tle1 phospholipase activity once bound to VgrG suggests that Tle1 dissociation from VgrG is required upon delivery.
History
DepositionAug 14, 2019-
Header (metadata) releaseApr 29, 2020-
Map releaseApr 29, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 8.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 8.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6sk0
  • Surface level: 8.1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6sk0
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10219.png

Downloads & links

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Map

FileDownload / File: emd_10219.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 1.024 Å
Density
Contour LevelBy EMDB: 8.1 / Movie #1: 8.1
Minimum - Maximum-26.706247 - 56.76904
Average (Standard dev.)-0.00000000000 (±1)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 358.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0241.0241.024
M x/y/z350350350
origin x/y/z0.0000.0000.000
length x/y/z358.400358.400358.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ350350350
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS350350350
D min/max/mean-26.70656.769-0.000

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Supplemental data

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Additional map: Masked sharpened map

Fileemd_10219_additional_1.map
AnnotationMasked sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_10219_additional_2.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map1

Fileemd_10219_half_map_1.map
AnnotationHalf map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map2

Fileemd_10219_half_map_2.map
AnnotationHalf map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tle1 effector bound to the VgrG spike of the type 6 secretion system

EntireName: Tle1 effector bound to the VgrG spike of the type 6 secretion system
Components
  • Complex: Tle1 effector bound to the VgrG spike of the type 6 secretion system
    • Protein or peptide: Putative type VI secretion proteinType VI secretion system

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Supramolecule #1: Tle1 effector bound to the VgrG spike of the type 6 secretion system

SupramoleculeName: Tle1 effector bound to the VgrG spike of the type 6 secretion system
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 956 KDa

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Macromolecule #1: Putative type VI secretion protein

MacromoleculeName: Putative type VI secretion protein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 92.657219 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNLTDSLQNV LSGVGTLNRY RLDIPSCPSS LDVEDFSGTE GISKIYRYDI IFTSTDRYPD AAWFLRKSAT LIMGTGLLES LTEQKKVHG VITDFRRISG SEDQAQYRIT LKPFISLLDK QFRTHRFFVN KSVPEVVEQI LTEHGLKGWE YEFSLKRTYP K REQINQYQ ...String:
MNLTDSLQNV LSGVGTLNRY RLDIPSCPSS LDVEDFSGTE GISKIYRYDI IFTSTDRYPD AAWFLRKSAT LIMGTGLLES LTEQKKVHG VITDFRRISG SEDQAQYRIT LKPFISLLDK QFRTHRFFVN KSVPEVVEQI LTEHGLKGWE YEFSLKRTYP K REQINQYQ ESDLRFIQRL LAEVGIFYFF TLHPDAQTEV IHFGDVQAAL IFDKTLPVNS PSGMSDSGTD SVWALNVEHR VV ESRVITN DYNHREAQNI LMSVPADMTR GEGYDTSYGE VYHYRPRHTE RGDKIDPAPE TANFWARLDH ERFLAEQTRI TGK STDASL LPAQVLTITD STPPVLPAPL QEPVLLTQLL FTGSRKSALQ VMLSAVPYSE IVCWRPPLLT RPKITGTMTA RVTS AKEGD IYAWQDASGM YRVKFDADRD DKNPGQESMP VRLAKPYSGD AYGFHFPLIQ GTEVAIAFEE GDPDRPYIAH ALHDS RHVD HVTDKNGTRN VIRTPANNKL RMEDKRGEEH IKLSTEYGGK TQLNLGHNVD ASRELRGEGA ELRTDDWISI RGGKGI FIS ADMQPQAQGK MLDMDEAIRQ LEQALSLARS MAKAATAANA TQGDISCQQR LNASLTDLTA PGMLLHAPDG IGMVSAR AL RIASGSESVG IMSGDNTDIT AGQSFTVVAE GAVSLLSRNQ GMQLLAAKGR VNIQAQSDDL SMSSQQNLDI QSSEGKVT V SANQELILAC GGAYIKLSGG NIELGCPGQI LLKSTNMQKM GPTSLDIASV EMPRGFGGGF ILTDEAGVPQ PSTPYRLTT AEGDILQGIT DENGKTAPVN TSIPSVVKVE FGKVKIHGET E

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.6 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.28 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 468438

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Atomic model buiding 1

RefinementProtocol: BACKBONE TRACE
Output model

PDB-6sk0:
The VgrG spike from the Type 6 secretion system

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